The present invention is in the field of biopesticides. More particularly, this invention relates to the finding that a novel strain of Bacillus pumilus, NRRL Accession Number B-30087, can inhibit a broad range of fungal plant diseases in vivo. The invention also relates to fungicidal compositions comprising this novel Bacillus strain, and the antibiotics and purified and non-purified fractions of this strain either alone, or in combination with other chemical and biological pesticides. The invention further relates to the synergistic fungicidal effect of using NRRL Accession No. B-30087 together with NRRL Accession No. B-21661, (CCRC 910106).
It is generally known that various microorganisms exhibit biological activity that are useful to control plant diseases. Although progress has been made in the field of identifying and developing biological pesticides for controlling various plant diseases of agronomic and horticultural importance, most of the pesticides in use are still synthetic compounds. Many of these chemical fungicides are classified as carcinogens by the Environmental Protection Agency (EPA), are toxic to wildlife and other non-target species. In addition, pathogens may develop resistance to chemical pesticides. See, e.g., Schwinn et al., in: Advances In Plant Pathology: Phytopathora infestans, The Cause of Late Blight of Potato, p. 244, Academic Press, San Diego, Calif. (1991).
Biological control offers an attractive alternative to synthetic chemical fungicides. Biopesticides (living organisms and the naturally produced compounds produced by these organisms) can be safer, more biodegradable, and less expensive to develop.
One commonly used biopesticide is the gram positive bacterium Bacillus thuringiensis. Pesticidal B. thuringiensis strains are known to produce crystal proteins during sporulation, which are specifically toxic to certain orders and species of insects and nematodes (See, e.g., U.S. Pat. No. 4,999,192 and U.S. Pat. No. 5,208,017). Proteinaceous endotoxins produced by B. thuringiensis also act as insecticidal agents against corn rootworm and other beetles (e.g., U.S. Pat. 5,187,09 and Johnson, T. J. et al. (1993), J. Econ. Entomol., 86:330-333). B. thuringiensis endotoxins have been shown to be effective as purified crystals, washed cell pellets, and expressed proteins. Warren et al. WO 96/10083, disclose non-endotoxin proteins produced during the vegetative stage of Bacillus cereus and B. thuringiensis. These vegetative proteins, called Vip1 and Vip2 have potent activity against corn rootworm (northern and western). See, Estruch et al. (1997), Nature-Biotechnology 15:137-141.
One B. thuringiensis thermostable metabolite, termed beta-exotoxin has also been shown to have pesticidal properties. Burgjeron and Biache (1979), Entomophaga 11:279-284, report a beta-exotoxin that is active against Colorado potato beetle (Leptinotarsa decemlineata). In addition, the known B. thuringiensis beta-exotoxins exhibit non-specific pesticidal activity, not only killing nematodes, but also flies, armyworm, mites, and corn rootworm. Sigma exotoxin has a structure similar to beta-exotoxin, and is active against Colorado potato beetle. See, Argauer et al. (1991), J. Entomol. Sci. 26: 206-213. Alpha-exotoxin is toxic to larvae of Musca domestica (Cluthy (1980), FEMS Microbiol. Lett. 8:1-7). Gamma-exotoxins are various proteolytic enzymes, chitinases and proteases. The toxic effects of gamma-exotoxins are only expressed in combination with beta-exotoxin or delta-endotoxin. See, Forsberg, C., xe2x80x9cBacillus thuringiensis: Its effects on Environmental Qualityxe2x80x9d National Research Council of Canada, Publication No. NRCC 15385, pp. 91-109 (1976). Stonard et al. (1994), ACS Symposium Series 551:25, report a water-soluble secondary metabolite active against corn rootworm in the supernatant of a Bacillus cereus strain.
Zwittermicin A is a water soluble, acid stable linear arninopolyol molecule (see, He et al. (1994), Tetrahedron Lett. 35(16):2499-2502) with broad-spectrum activity against many fungal and bacterial plant pathogens. Zwittermicin A is also known to enhance the activity of B. thuringiensis. Manker et al. (WO 96/39037) were the first to determine the B. thuringiensis-enhancing abilities and properties of zwittermicin A. Subsequently, Schnepf et al. also reported that zwittermicin A enhanced B. thuringiensis (U.S. Pat. No. 5,702,703).
Bacilli are known to produce antifungal and antibacterial secondary metabolites. See, Korzybski et al. xe2x80x9cAntibiotics isolated from the genus Bacillus (Bacillaceae)xe2x80x9d in: Antibioticsxe2x80x94Origin, Nature and Properties, American Society for Microbiology, Washington, D.C. Vol. III (1978), and Berdy, CRC Handbook of Antibiotic Compounds, Vols. I-XIV, CRC Press, Inc., Boca Raton, Fla. (1980-87). Compounds produced by B. pumilus include micrococcin P, pumilin, and tetain.
Kawaguchi et al., in U.S. Pat. No. 4,250,170, isolated a novel water-soluble antibiotic from Bacillus with activity against a broad range of gram positive and gram negative bacteria. Stabb et al. (1990) Applied Environ. Microbiol. 60:4404-4412, have identified certain Bacillus spp. (Bacillus spp. include B. subtilis, B. cereus, B. mycoides, B. thuringiensis) strains that exhibit antifungal activity. These strains have been shown to produce zwittermicin A and/or kanosamine. See, Milner et al., Appl. Environ. Microb. 62:3061-3066 (1996). These are antibiotic agents that are effective against the soil borne disease damping off, caused by Phytopathora medicaginis, P. nicotianae, P. aphanidermatum or Sclerotinia minor (See, Stabb et al., supra). Zwittermicin-A is a water soluble, acid stable linear aminopolyol molecule. See, He et al., (1994) Tetrahedron Lett. 35(16):2499-2502. It has broad spectrum activity against many fungal and bacterial plant pathogens. Kanosamine (Milner et al., 1996) also inhibits a broad range of fungal plant pathogens and a few bacterial species.
Handelsman et al., in U.S. Pat. No. 5,049,379, describe how Zwittermicin A-producing B. cereus controls damping off in alfalfa and soybeans. When the seed was coated with B. cereus ATCC 53522, the pathogenic activity of root rot fungus was inhibited. Similarly, application of spore-based formulations of certain B cereus strains to soybean seeds or the soil surrounding the seeds has been shown to improve soybean yield at field sites. See, Osburne et al. (1995) Am. Phytopathol. Soc. 79(6):551-556. Methods of applying biopesticides are well known in the art and include, for example, wettable powders, dry flowables, microencapsulation, and liquid formulations of the microbe, whole broth or antibiotic fractions from suitable cultures. See, e.g., U.S. Pat. No. 5,061,495 to Rossall and U.S. Pat. No. 5,049,379 to Handelsman et al.
Tsuno et al. (1986) J. Antibiotics XXXIX(7):1001-1003, report on a new amino sugar antibiotic from B. pumilus with activity against a broad range of bacteria in vitro.
Khmel, I. A. et al., (1995) in SU 1817875 disclose a novel strain of Bacillus pumilus VKM CR-333D, which is used to control fungal phytopathogens and bacteria.
Leifert et al., J. Appl. Bacteriol. 78:97-108 (1995), report the production of anti-Botrytis and anti-Alternaria antibiotics by two Bacillus strains, B. subtilis CL27 and B. pumilus CL 45. The whole broth and cell-free filtrates are active against Botrytis and Alternaria in in vitro tests and are active against Botrytis in in vivo small plant tests on Astilbe. Leifert et al. (1997) U.S. Pat. No. 5,597,565 disclose B. subtilis, B. pumilus, and B. polymyxa that are particularly effective at inhibiting post harvest disease causing fungi, Alternaria brassicicola and Botrytis cinerea. They also disclose the presence of antibiotics produced in the cell-free culture filtrate and their activity at different pH values, but they do not identify these compounds. The compounds from B. subtilis lose activity at low pH, while the activity from the B. pumilus extracts occurs only at pH values below 5.6. Leifert et al. (1998) U.S. Pat. No. 5,780,080 disclose cabbages that can be treated with B subtilis, B pumilus, and B. polymyxa strains to inhibit Alternaria brassicicola and Botrytis cinerea. 
Loeffler et al. (1986) J. Phytopathology 115:204-213, disclose B. subtilis, B. pumilus, B. licheniformis, and B. coagulans strains that produce various antibiotics with antifungal and antibacterial activity. B. pumilus produced bacilysin and iturin A. Bacilysin is a very small compound with a molecular weight of 270 that inhibits only yeast. The iturins, which are soluble in polar solvents, have broad antifungal and antibacterial activity.
In U.S. Pat. No. 5,344,647, Rossall discloses Bacillus subtilis strains with broad anti-fungal activity. Additionally, U.S. Pat. No. 5,061,495 to Rossall, provides a novel antibiotic from B. subtilis that is 63,500 daltons, precipitates at a pH below 5 and has activity against gram positive bacteria and fungi (Botrytis and Erysiphe). Sholberg et al. (1995) Can. J. Microbiol. 41:247-252, Swinburne et al. (1975) Trans. Brit. Mycol. Soc. 65:211-217, Singh and Deverall, (1984) Trans. Br. Mycol. Soc. 83:487-490, Ferreira et al. (1991) Phytopathology 81:283-287 and Baker et al. (1983) Phytopathology 73:1148-1152. All disclose the use of Bacillus spp. and Bacillus subtilis as biocontrol agents of fungal plant pathogens. Pusey et al. (1988) Plant Dis. 72:622-626, Pusey et al., U.S. Pat. No. 5,047,239, and McKeen et al. (1986) Phytopathology 76:136-139 disclose control of post harvest fruit rot using B. subtilis. McKeen et al., supra, have shown that antibiotics similar to the low molecular weight iturin cyclic polypeptides contribute to this fungicidal activity of B. subtilis. 
Liu et al., in U.S. Pat. No. 5,403,583 disclose a Bacillus sp., (ATCC 55000) and a method to control the fungal plant pathogen, Rhizoctonia solani. Islam and Nandi (1985) J. Plant Dis. Protect. 92(3):241-246, disclose a Bacillus sp. with antagonism to Drechslera oryzae, the causal agent of rice brown spot. The same authors, Islam and Nandi (1985) J. Plant Dis. Protect. 92(3):233-240, also disclose in-vitro antagonism of Bacillus sp. against Drechslera oryzae, Alternaria alternata and Fusarium roseum. They discuss three components in the culture filtrate. The most active antibiotic was highly soluble in water and methanol with a UV peak at 255 nm and a shoulder at 260 nm that proved to be a polyoxin-like lipopeptide. Cook et al. (1987) Beltwide Cotton Production Research Conferences, Dallas, Tex., pp. 43-45, disclose the use of a suspension of Bacillus sp. to reduce the number of cotton plants killed by Phymatotrichum omnivorum, a cause of cotton root rot.
B""Chir and Namouchi (1988) Revue Nxc3xa9matologique 11(2):263-266, report on a Bacillus pumilus that stimulates nematode trapping fungi to increase their ability to trap nematodes. B""Chir and Belkadhi (1986) Med. Fac. Landbouww. Ryiksuniv. Gent 51/3b:1295-1310, discuss the cellular interactions of a fungus (Fusarium) and nematodes that cause infection in citrus. The fungus is associated with B. pumilus (they occur together) and when the nematode is also there, the fungus is more severe. B. pumilus appears to be providing food for the nematodes. Gokte and Swarup (1988) Indian J. Nematol. 18(2):313-318, report on B. pumilus that are nematicidal, but they do not report any antifungal activity. Slabospitskaya et al. (1992) Mikrobiol Zh (Kiev) 54(6):16-22, compare many different Bacillus, including B. pumilus for their ability to produce chitinases, but they report no activity on plant pathogens. The B. pumilus produce the lowest chitinase levels. Mclnroy et al. (1995) Plant and Soil 173(2):337-342, did a survey of the many types of bacteria, including many Bacillus and B. pumilus that are endophytes within plant stems and roots. However, they show no evidence that these endophytic strains are antifungal. Chemin et al. (1995) Molecular Genetics, found a Bacillus pumilus that has a wide spectrum of activity against bacteria (e.g., Xanthomonas, Pseudomonas, Erwinia) and fungi that cause plant disease. Fey et al. (1991) Akad Landwirts Kart, report on B. pumilus strains that provide seed potatoes some protection from Rhizoctonia solani. 
A novel antibiotic-producing Bacillus sp. is provided that exhibits antifungal activity only on certain specific plant pathogens and no antibacterial activity. Also provided is a method of treating or protecting plants, fruit and roots from fungal infections comprising the step of applying an effective amount of an antibiotic-producing Bacillus sp. The antibiotic-producing Bacillus sp. can be provided as a suspension in a whole broth culture or as a partially-purified antibiotic-containing supernatant obtained from a whole broth culture of an antibiotic-producing Bacillus sp. Also provided is a novel water-soluble antibiotic that exhibits specific antifungal activity and no antibacterial activity.
The present invention also provides a novel compound that enhances the insecticidal activity of B. thuringiensis. The compound is isolated from whole broth culture or supernatant of B. pumilus, that when combined with B. thuringiensis, enhances its insecticidal activity. The invention also includes methods of treating plants to control insect infestations on or in plants with a bacterial suspension of a Bacillus or a metabolite-containing supernatant of a culture of a Bacillus or purified metabolites.
The present invention further provides combining strain B-30087 with strain B-21661 (AQ 713) for use as fungicides, where the use of the strains together provides a greater efficacy than if either were used alone.